Lesser Periwinkle (Vinca minor L.), a member of Apocynaceae, is not only an ornamental plant with lilac-blue flowers, but also a medical plant producing an important alkaloid, vincamine, found in the leaves which shows a pronounced cerebrovasodilatory and neuroprotective activity. This plant is native to northern Spain, western France, central and southern Europe, and Caucasus. It has been recently cultivated for pharmaceutical purposes by Zardband Botanical Garden in Iran. Since the quality of herb material and alkaloid concentration is greatly influenced by environmental conditions, in this study, we report the isolation and identification of major alkaloids along with the quantification of vincamine as the pharmacologically most important component. Alkaloids from the aerial parts of V. minor were isolated and purified using different chromatographic methods. The structures of these alkaloids were determined on the basis of their physical and spectroscopic data. The concentration of vincamine was determined by high performance liquid chromatography using Tracer Excel 120 ODS A C18 column. Five indole alkaloids including vincaminorine, vincaminoreine, minovine, minovincine, and vincamine (Figure 1) were isolated from the aerial parts of V. minor. Vincamine was found to be the dominant alkaloid in this plant with the content of 0.057% of the dried plant mass. This plant may be used as a natural source for pharmaceutical purposes in Iran, due to the presence of biologically active alkaloids especially vincamine as the major alkaloid in Lesser Periwinkle cultivated.